Various aspects of protein degradation will be studied using red cell-mediated microinjection of proteins into cultured mammalian cells. We will determine whether dissimilar subunits of proteins turn over at identical rates and whether covalent-binding of substrate analogs to enzymes retards their degradation. We will attempt to detect initial reactions in protein degradation by coinjection of protease inhibitors and labeled proteins or by exposing injected cells to low temperature. To determine whether lysosomes are involved in the degradation of soluble proteins we will couple nondegradable D-amino acid peptides to proteins prior to injection. Non-degraded peptides are expected to accumulate within the lysosomes if degradation of protein occurs there. We also propose to isolate mutant HeLa cells that degrade immunoglobin G at reduced rates.